This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Copper is required for the activity of many enzymes involved in respiration, neuron function, formation of connective tissue, endocrine processes, and radical detoxification in the human body. Wilson disease ATPase and Menkes disease ATPase regulate copper concentration in the cell and deliver copper to biosynthetic pathways. These proteins are targets of many mutations that cause severe metabolic disorders. Importantly, Wilson and Menkes ATPases are involved in cancer resistance to platinum- based chemotherapeutic drugs. To understand the critical steps in the activity cycle of the copper ATPases, we will investigate the structure, molecular motions and interactions of the isolated domains, or functional modules, of the Wilson and Menkes proteins using multidimensional Nuclear Magnetic Resonance (NMR), a technique uniquely suited for studying protein structure and dynamics in solution. We will trace the sequence of molecular events involved in substrate binding by copper-transporting ATPases and analyze the structural basis of several frequent disease causing mutations. To trace the pathway of copper in Wilson and Menkes disease ATPases, we will attempt to map the copper-binding site in the cell membrane. This work is expected to improve the understanding of an important class of transport proteins and provide a new insight into the molecular basis of the disorders of copper metabolism. Structures of the individual domains of Wilson and Menkes ATPase will facilitate design of the new inhibitors and modulators of these enzymes, which may help to overcome certain types of drug resistance in cancer.